检测纽布病毒的Real-time RT-PCR方法的建立与应用

doi:10.11843/j.issn.0366-6964.2019.04.023

Abstract

Abstract:

Nebovirus (NeV) is an emerging causative agent of calf diarrhea in China, the aim of the study was to establish a real-time RT-PCR assay for detecting NeV. The TB Green real-time RT-PCR assay was successfully established through designing primers targeted to RdRp fragment of NeV strains in China and optimizing the reaction conditions and system. The Real-time RT-PCR assay showed a good linear relationship between 2.9×10¹ and 2.9×10⁹ copies·μL^-1, linear correlation coefficient R²=0.999 4, and amplification efficiency was 98%. This method only specifically detected NeV, and did not detect other unrelated pathogens; the minimum detection limit was 29 copies·μL^-1; the intra-and inter-coefficients of variation were 0.89%-1.89% and 0.83%-1.15%, respectively; comparing to other three reported RT-PCR assays, the assay in this study has a significant high detection rate for NeV in clinical samples (P<0.05). In 135 diarrhea samples of dairy cows from Xinjiang and Liaoing regions from August to November 2018, the NeV detection rate was 67.41%, and the farms positive rate was 100% (15/15). The assay for detecting NeV established in this study has a good specificity and stability, which provide an effective means for detection and epidemiological investigation of NeV.

Key words: nebovirus, real-time RT-PCR, calf diarrhea, detection

CLC Number:

S852.659.6

GUO Zijing, HE Qifu, TANG Cheng, ZHANG Bin, YUE Hua. Establishment and Application of a Real-time RT-PCR Assay for Detecting Bovine Nebovirus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(4): 893-900.

References

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Establishment and Application of a Real-time RT-PCR Assay for Detecting Bovine Nebovirus

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